Actinotrichia contribute to the pectoral breeding tubercle cluster phenotype required for successful zebrafish reproduction. (A) Percentage of wild-type eggs (n=6 females) fertilized by wild-type (n=3 males) and double homozygous (n=3 males) sperm by IVF. Data pooled from two trials of 3 wild-type male×wild-type female and 3 and1−/−and−/− male×wild-type female crosses. Data are mean±s.e.m. Dots represent individual data points. (B) Stills from overhead views of courtship videos of wild-type and double mutant males with wild-type females in Movie 1. Double mutant stills were taken from courtship sequence 1. Body contortions are emphasized at dorsal midlines (males, orange dashed lines; females, white dashed lines). (C) Pectoral breeding tubercle cluster phenotype in wild-type [n=48 rays with breeding tubercles (BTs)] and double mutant (n=44 rays with BTs) males. Left: whole pectoral fins show the arrangement of BT clusters relative to ray phenotype. Brackets indicate BT clusters. Scale bars: 1 mm. Right: magnified views of the whole fin. Wild-type BT clusters are found on anterior-central pectoral fin rays. Double homozygous BT clusters present with a range of defects, appearing normal (n=11/44), reduced (n=25/44) or segmented (n=8/44) in different fin rays of different individuals. Scale bars: 500 µm. (D) Mallory-stained transverse cryosections of wild-type (n=32 sections, 3 males) and double homozygous (n=30 sections, 3 males) pectoral BT clusters. Double mutant clusters contained 3 or 4 rows of BTs (n=12/30), comparable to wild-type clusters, but also often had 1 or 2 rows of BTs (n=18/30). Scale bars: 100 µm. (E) Whole-mount Mallory staining of all fins and jaw in wild-type (n=4) and double mutant (n=3) males. BTs found along the dorsal, anal and pelvic fin rays are stained darker than their periphery. BTs arranged in dense clusters in the pectoral fin and the jaw are stained red. Orange arrowheads indicate individual BTs; orange brackets indicate BT clusters. Scale bars: 250 µm.
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